The disintegrin metalloprotease (or ADAM) family of cell surface proteolytic enzymes is known to play roles in sperm-egg binding and fusion, muscle cell fusion, neurogenesis, modulation of Notch receptor and ligand processing, and processing of the pro-inflammatory cytokine, TNFα (Primakoff and Myles, Trends Genet 16:83-87, 2000). The ADAMs have been shown to consist of pre-, pro-, protease, disintegrin-like-, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains. Members of a novel sub-family of the ADAMs, the ADAMTS proteins, lack the transmembrane domain and contain unique thrombospondin motifs, believed to mediate their binding to the extracellular matrix (Tang and Hong, FEBS Lett. 445:223-225, 1999). Two members of the ADAMTS family, namely ADAMTS-4 and -5 (also referred to as ADAMTS-11), have been shown to be capable of aggrecan cleavage. Aggrecan is the major proteoglycan of cartilage (Abbaszade et al., J. Biol. Chem. 274:23443-23450, 1999; Tortorella et al., Science 284:1664-1666, 1999). As a result, these proteins have been implicated in the cartilage damage associated with osteoarthritis and inflammatory joint disease, and have been named “aggrecanase-1” (Genbank Accession NM 005099) and “aggrecanase-2” (Genbank NM 007038), respectively.
Aggrecanases and matrix metallo proetinases (MMPs) have been shown to cleave aggrecan at a number of different sites (Pratta et al., J. Biol. Chem. 275:39096-39102, 2000; Sandy et al., Biochem. J. 351:161-166, 2000; Tortorella et al., J. Biol. Chem. 275:18566-18573, 2000). Products resulting from cleavage of aggrecan at the site Glu373-Ala374, in the interglobular domain of aggrecan, have been shown to accumulate in synovial fluid of patients with osteoarthritis and inflammatory joint disease (Lohmander et al., Arthritis Rheum. 36:1214-22, 1993). Aggrecanase-1 and -2, but not MMPs, are able to cleave aggrecan at this site. A 40 amino acid peptide representing the sequence of aggrecan surrounding the aggrecanase cleavage site (PCT Publication Number WO 00/05256) was able to serve as a substrate for aggrecanase enzymatic activity; however, no peptides less than 40 amino acids in length functioned as suitable substrates for aggrecanase activity, suggesting that shorter substrates, such as substrates of 20 amino acids in length, would not work. Minimum size limits for aggrecanase substrates are consistent with studies suggesting that aggrecanase activity is sensitive to the amino terminal truncation of aggrecan (Horber et al., Matrix Biol. 19:533-543, 2000). Glycosylation of the aggrecan substrate has also been shown to affect aggrecanase activity (Pratta et al., J. Biol. Chem. 275:39096-39012, 2000).
A sensitive and specific assay for the aggregan degrading metalloproteases, suitable for high-throughput screening, would be helpful in identifying inhibitors of these enzymes for potential therapeutic agents against cartilage damage associated with osteoarthritis and inflammatory joint disease. This invention relates to amino acid peptides shorter than 40 amino acids, unrelated to the aggrecan sequence, but containing aggrecanase sensitive sites, and their use in assays suitable for high throughput screening (HTS) formats.